Treatment of HIV-associated dysmorphia/dysmetabolic syndrome (HADDS) with or without lipodystrophy

ABSTRACT

Pathological regional adipose tissue accumulation associated with HIV-associated dysmorphic/dysmetabolic syndrome (HADDS) which may occur with or without subcutaneous adipose tissue lipodystrophy (and which is also described as HIV-associated adipose redistribution syndrome or HARS and other specific medical terms), is treated by administering an effective amount of human growth hormone or other substance which binds to and initiates signalling of the hGH receptor. Alternatively, a substance which stimulates production of endogenous hGH, such as human growth hormone releasing hormone, may be administered. HADDS and related syndromes include abnormal adipose tissue accumulation in the visceral, submandibular, supraclavicular, pectoral, mammary and/or dorsocervical (buffalo hump) area, and/or with subcutaneous lipomas, with or without associated metabolic or other physiologic abnormalities.

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the benefit of provisional applicationNo. 60/114,389, filed Dec. 30, 1998, the entire contents of which beinghereby incorporated herein by reference.

FIELD OF THE INVENTION

The present invention is directed to a method for treatingHIV-associated dysmorphia/dysmetabolic syndrome or HIV-associatedadipose redistribution syndrome, and related syndromes.

BACKGROUND OF THE INVENTION

Advances in antiviral treatment of HIV infection, along withdevelopments in the prophylaxis and therapy of opportunistic infections,have greatly improved the long-term health of HIV-infected individuals.However, along with improved antiretroviral therapy a new syndrome hasdeveloped, which is identified herein as HIV-associateddysmorphia/dysmetabolic syndrome, or HIV-associated adiposeredistribution syndrome (HARS), or hereafter abbreviated HADDS.

HADDS involves pathological accumulation of adipose tissue in specificregional depots. The pathologic adipose tissue accumulation of HADDS mayalso be associated with abnormal adipose tissue depletion elsewhere(lipodystrophy or lipoatrophy), with or without associated metabolicabnormalities, premature atherosclerotic lesions, depletion of lean bodymass, and/or other abnormal physiology.

This recently discovered clinical disorder, which has been referred toby a number of terms, including HIV lipodystrophy syndrome and otherterminology, has important public health consequences, as describedfurther below.

HADDS patients typically present with abnormal accumulation of adiposetissue in the abdomen, specifically in the visceral adipose tissuecompartment (Miller et al, 1998, Kotler et al, 1999; Engelson et al,1999). HADDS patients may also present with abnormal adipose tissueaccumulation in the dorsocervical area (“buffalo hump”), thesubmandibular area (“horse collar”), the pectoral, mammary, and/orsupraclavicular areas, and/or with subcutaneous lipomas (encapsulatedbenign fatty tumors, single or multiple).

Abnormal, involuntary, pathological, and often dysmorphic accumulationof adipose tissue is sufficient to diagnose a HIV-infected patient withHADDS. However, in addition to developing abnormal adipose accumulation,some HADDS patients develop abnormally depleted subcutaneous adiposetissue, termed “peripheral lipodystrophy” (or lipoatrophy) at otherspecific sites. This adipose depletion is typically observed in the face(buccal, parotid, and periauricular fat pads), and in the subcutaneousadipose tissue surrounding the limbs, trunk, and/or gluteal regions.Also, some HADDS patients may present with metabolic abnormalities (Carret al, 1998a, 1998b, 1999a, 1999b; Lipodystrophy Rapid Report, 1999).

The metabolic abnormalities associated with HADDS and lipodystrophysyndrome typically involve disordered lipid and glucose metabolism.Clinical manifestations may include fasting hypertriglyceridemia,hyperlipidemia, and abnormalities of the insulin/glucose axis (elevatedfasting insulin, elevated C-peptide, insulin resistance or reducedinsulin sensitivity), with or without overt diabetes (Carr et al, 1998a,1998b, 1999a, 1999b; Henry et al, 1998; Grunfeld, 1999).

Abnormal adipose tissue accumulation abnormalities occur more frequentlyin female than male cases diagnosed with HADDS, while fat depletion,hyperglycemia, and hyperlipidemia are more commonly observed in malecases (Muurahainen, 1999). Hence the presentation of HADDS appears tovary by gender.

There are preliminary reports suggesting that patients with HADDSexhibit preclinical evidence of increased risk for coronary heartdisease (CHD). Preclinical indicators of CHD include increased coronaryartery calcification (CAC) as quantified by electron beam computedtomography (EBCT), and extracoronary indicators such as increased intimamedia thickening (IMT) in the carotid artery and impaired bloodflow-mediated dilation in the brachial artery, as quantified byultrasonography, which signify endothelial dysfunction that may lead toatherosclerosis and CHD. In eight patients with HADDS who developedincreased abnormal girth with abnormally accumulated visceral adiposetissue after initiation of HIV rotease inhibitor (PI) therapy whounderwent EBCT, Kosmiski et al (1999) reported a mean CAC scoreconsistent with minimal identifiable plaque burden. There are alsopreliminary reports indicating that HIV patients receiving PIs displayabnormal carotid IMT (Maggi et al, 1999) and impaired brachialflow-mediated dilation (Stein, 1999), signifying endothelialdysfunction. However, it is unclear which, if any, of the patients inthe two aforementioned preliminary reports were HADDS patients, and whatpercentage of HADDS patients have preclinical indicators of abnormalendothelial function.

There have also been case reports of premature coronary artery diseasein HIV patients under age forty who have been receiving combinationantiretroviral therapy that include a protease inhibitor (Henry et al,1998). However, it may take years before well-designed, prospective,observational cohort studies precisely quantify the risk of prematurecoronary artery disease in patients with HADDS.

Some patients with HADDS also exhibit involuntary weight loss withdepletion of lean body mass (AIDS wasting or cachexia), and possiblydepletion of lean body mass without overt weight loss (occult wasting)(Muurahainen, unpublished observations, data on file, SeronoLaboratories, Norwell, Mass.).

Other abnormal physiology that may be observed in patients with HADDS orlipodystrophy syndrome include gout and pancreatitis (presumablyresulting from severe hypertriglyceridemia), hepatic steatosis (possiblyreflecting chronic lactic acidosis), hypogonadism, and possibly otherhormonal abnormalities (Henry et al, 1998; Brinkman, 1999; LipodystrophyRapid Report, 1999).

HADDS and lipodystrophy syndrome may or may not be associated with otherabnormalities, such as cutaneous abnormalities (such as thinning hair,hair loss, hair brittleness, dry skin, abnormal nails, ingrowntoenails), disorders of the coagulation syndrome that result inincreased bleeding in hemophiliacs, osteoporosis or avascular necrosisof the hips, peripheral neuropathy, nausea, fatigue, weight loss,chronic diarrhea, fever, mennorhagia and menstrual abnormalities,impaired sexual dysfunction (decreased libido, erectile dysfunction),and abnormalities of the genitalia resembling Peyronie's Disease (Carret al, 1998a, 1998b, 1999a, 1999b; Lipodystrophy Rapid Report, 1999).

In sum, HADDS is a recently discovered multisystemic, gender dimorphicdisorder associated with HIV infection that includes (1) regionalchanges in adipose distribution, frequently dysmorphic, that result fromabnormal regional accumulation of adipose tissue, with or withoutlipodystrophy, (2) occasionally observed in conjunction withabnormalities of lipid and glucose metabolism, and (3) possiblyassociated with other physiologic abnormalities, including prematureatherosclerotic lesions, depletion of lean body mass, and otherabnormalities.

Since HADDS and HIV-associated lipodystrophy syndrome have only recentlybeen described, standardized nomenclature or consensus definition(s) forthese syndromes are lacking. Kotler and Schambelan (1999) predict thatwe may eventually see an official case definition that has major andminor criteria, similar to what is already in place for rheumaticdisorders such as systemic lupus erythematosis (Kotler and Schambelan,1999).

In recent years, a plethora of terms and nomenclature have been used byscientists, clinicians, and patient advocates to describe the syndromeand its manifestations. Any of the manifestations may be observed inmen, women, and children with HIV infection who develop the syndrome.The terms describing it may be found in the peer-reviewed scientificliterature, posted in discussions and reviews on the internet. The termsprovided below are non-inclusive. New terms are continually added.

Terms that have been used to describe the syndrome or subsets of itinclude: HIV-associated dysmorphia/dysmetabolic syndrome (HADDS);HIV-associated adipose redistribution syndrome (HARS); lipodystrophysyndrome and HIV-associated lipodystrophy syndrome (HALS); HIV-relatedperipheral lipodystrophy (HIPL); HIV-associated fat redistributionsyndrome (HIVFRES) or fat redistribution syndrome (FRS); fatmaldistribution syndrome (FMS), HIV-associated dysmorphia/metabolicSyndrome (HADMS), abnormal body fat (ABF) accumulation, and proteaseinhibitor-associated lipodystrophy (PI-AL).

Terms used to describe abnormal accumulation of abdominal adipose tissuein patients with the syndrome include: HIV-associated “Crix belly” and“protease paunch” (although the truncal adiposity may not be directlyrelated to Crixivan or other protease inhibitors); “pouch belly”;truncal adiposity, truncal obesity, central obesity, abdominaladiposity, increased waist to hip ratio (WHR), increased waist-to-thighcircumference ratio (by anthropometry), increased truncal to limb fatratio (by DXA scan), increased abdominal visceral adipose tissue (VAT)with decreased subcutaneous adipose tissue (SAT) and increased VAT/SATratio by CT or MRI scan, and “Pseudo-Cushing's syndrome”.

Terms used to describe other types of fat accumulation in patients withHADDS include HIV-related buffalo hump, abnormal accumulation ofdorsocervical fat, “dorsocervical lipodystrophy” (a misnomer because itimplies depletion of the dorsocervical fat pad), increased neck fat,facial fat accumulation, double chin, moon face, submandibular fataccumulation (“horse collar”), supraclavicular fat pad accumulation,multiple symmetric lipomatosis, “lumps and bumps”, Madelung's syndrome,HIV-related breast enlargement, mammary fat hyperplasia andgynecomastia. (Note that it is unclear if there is true gynecomastia, orwhether there is hypertrophy of subcutaneous chest fat other thanmammary tissue), “chest fat accumulation”, and peripheral adiposity.

Terms used to describe abnormal depletion of adipose tissue found insome patients with HADDS and lipodystrophy syndrome include:pseudocachexia, peripheral lipodystrophy, pure lipoatrophy, “Lipo”,facial wasting, facial wrinkling, sunken cheeks, sunken eyes, templehollowness, prominent zygomatic arch, “cadaveric facies”, buccal,parotid, and periauricular fat pad wasting, limb wasting, skinny, stickarms and stick legs with symmetrical, prominent non-varicose veins,muscularity and bones, butt wasting, saggy buttocks with loose skinfolds, loss of buttock fat contour, and hollowing of the buttocks.

For the purpose of the invention described herein, a distinction is madebetween HADDS and HIV lipodystrophy syndrome. Several types ofHIV-related lipodystrophy syndrome(s) have been described: patientspresenting only with adipose tissue accumulation, those presentingadipose accumulation and depletion (“mixed syndrome”), and thosepresenting only with adipose tissue depletion (“pure lipoatrophy”)(Rosenbaum, 1999; Saint-Marc, 1999; Thiebaut et al, 1999; Viard, 1999).

For the purpose of the invention described herein, HADDS patients areconsidered to be a subset of patients with HIV-related lipodystrophysyndrome who exhibit abnormal adipose tissue accumulation. HADDSpatients are lipodystrophy patients who exhibit either adiposeaccumulation alone or adipose accumulation associated with adiposedepletion elsewhere (“mixed syndrome”). The rationale for focusing onHADDS patients as a clinically important subset of patients withlipodystrophy syndrome, is that patients with abnormally accumulatedadipose tissue are the ones for whom the invention, a lipolytic(adipose-depleting) agent, will be recommended as a treatment, incontrast to patients with lipodystrophy syndrome who exhibit onlyadipose tissue depletion, or pure lipoatrophy.

For purposes of the invention described herein, it makes sense from ascientific perspective to focus on the treatment of pathologicallyaccumulated adipose tissue with associated metabolic and physiologicabnormalities, rather than to focus on treatments for depleted adiposetissue. As discussed further below, excess accumulation of visceraladipose tissue has been associated with adverse clinical outcomes. Thelong term clinical consequences of adipose tissue depletion alone (purelipoatrophy per se), if any, are presently unknown.

Since 1997, the unusual and unexpected morphologic features thatcomprise HADDS and HIV-related lipodystrophy syndrome have beenincreasingly observed and reported. Manifestations of the syndrome werefirst observed in conjunction with use of highly active combinationantiretroviral therapies that included an HIV protease-inhibiting agent(Carr et al, 1998a, 1998b, 1999a, 1999b; Lo et al, 1998), but themanifestations have also been observed in HIV patients who have neverreceived protease inhibitors (Lo et al, 1998; Kotler, 1998; Carr et al,1999a, 1999b; Mercie et al, 1999; Gervasconi, 1999; Hadigan et al, 1999;Brinkman, 1999).

Abnormal adipose tissue accumulation, the primary pathologic lesion ofHADDS may develop rapidly (within weeks to months after initiating a newantiretroviral regimen) or may develop gradually over periods of one toseveral years. The etiology of the syndrome is unknown. It is unclearwhether the HADDS and lipodystrophy syndromes are drug-related, related,and if they are drug-related, what antiretroviral agents or classes ofagents are causal. Both HIV-1 protease inhibitors and HIV-1 reversetranscriptase inhibitors have been proposed as etiologic agents (Carr etal, 1998a, 1998b, 1999a, 1999b; Saint-Marc, 1999; Brinkman, 1999).

Although manifestations of HADDS and lipodystrophy syndrome are moreprevalent in HIV/AIDS patients who have received combinationantiretroviral therapies (especially highly active antiretroviraltherapy with protease inhibitors), in some cases the syndrome or some ofits manifestations have been observed in patients who have neverreceived antiretrovirals (Lo et al, 1998; Carr et al, 1998a, 1998b,1999a, 1999b; Mercie et al, 1999).

Manifestations of the syndrome sometimes partially abate whenantiretroviral medications are discontinued or changed, but themanifestations often do not abate completely or resolve relatively formany months or up until a year after the switch in therapy (Saint-Marc,1999; Rosenbaum, 1999; Ruiz, 1999; Moyle; 1999, Gatell; 1999). Thisraises a concern that the syndrome might occur in conjunction withimmune reconstitution or autoimmune phenomena associated with chronicsuppression of HIV-1 (Kotler, 1998).

As mentioned previously, for a number of reasons the dysmorphic bodilychanges and metabolic manifestations of HADDS are of important publichealth significance. Abnormal adipose accumulation becomes clinicallysignificant when it results in physical discomfort or disability due toa variety of symptoms, including the following types of symptoms:headaches and inability to fully extend the neck due to buffalo hump;abdominal cramping, indigestion, constipation, shortness of breath andrespiratory insufficiency, or ventral hernias due to rapid accumulationof excess visceral adipose tissue. Abnormal accumulation of visceraladipose tissue is also a significant independent risk factor forcardiovascular disease.

The physical manifestations of HADDS are also of public healthsignificance because these manifestations are perceived by many patientsto be disturbing, disfiguring, appalling, stigmatizing, and/orthreatening loss of privacy. Some patients with the syndrome developbody image disturbance, depression, and agoraphobia (Cheng, 1999; Forumfor HIV Collaborative Research, 1999). The unusual and increasinglywell-recognized physical characteristics of HADDS such as buffalo hump,truncal obesity, breast enlargement, horse collar, supraclavicular fatpads, facial and limb wasting, and lipomas have the potential to becomesocially stigmatizing because these recognizable features instantlyreveal that a patient probably has HIV/AIDS (“a forced form of HIVidentification”, according to Cheng (1999), as cited in LipodystrophyRapid Report (1999)).

Concerns about health, appearance, potential stigmatization and loss ofprivacy in the workplace and elsewhere, may lead some patients withHADDS to discontinue otherwise effective antiretroviral therapy (Strubleand Piscitelli, 1999). These concerns may also lead other HIV patientsto refuse to initiate medically necessary antiretroviral therapies forfear of developing the syndrome and the increased medical risksassociated with it (Cheng, 1999). This could eventually lead toincreased HIV infectivity, more opportunistic complications, andincreased medical costs.

The increased cardiovascular risk factors associated with HADDS are alsoof important public health significance. These risk factors includeabnormally accumulation of visceral adipose tissue, hyperlipidemia,hyperinsulinemia, and premature atherosclerotic changes associated inHADDS, may result in increased risk for cardiovascular disease andstroke (Henry et al, 1998).

Other metabolic manifestations associated with HADDS syndrome also havethe potential to become medically significant problems warrantingtherapeutic intervention. These metabolic manifestations include sudden,severe hyperglycemia, new-onset diabetes mellitus, pancreatitis and goutdue to pronounced hypertriglyceridemia, and difficult-to-managehyperlipidemia that may include elevated total cholesterol, elevated lowdensity lipoprotein (LDL) cholesterol, and decreased high densitylipoprotein (HDL) cholesterol, and other lipoprotein abnormalitiesassociated with increased cardiovascular risk (Henry et al, 1998).

With respect the invention described herein, it is worth noting thatantiretroviral agents (especially HIV protease inhibitors) undergosubstantial hepatic metabolism. It is difficult to pharmacologicallymanage hyperlipidemia and hyperinsulinemia in HIV patients receivingcombination antiretroviral therapy because the antiretroviral agentstypically interact with anti-hyperlipidemics (statins, fibrates) andanti-hyperglycemic agents (thiazides and glitazones), which also undergosignificant hepatic metabolism (Henry et al, 1998). A therapy such asthe invention described herein, using an agent which is not primarilymetabolized by the liver, and which has the potential to reducehyperlipidemia and improves insulin sensitivity while depletingabnormally accumulated visceral adipose tissue, may be of special valuein the treatment of HADDS.

Moreover, other physiologic abnormalities that have been suggested to beevaluated as part of research to develop a case definition oflipodystrophy syndrome, which may ultimately be associated with HADDS,may warrant therapeutic intervention (Lipodystrophy Rapid Report, 1999).These abnormalities include hypertension, hypogonadism, impaired sexualfunction (decreased, erectile dysfunction), menstrual irregularities,hair loss, dry skin, and ingrown toenails that can become infected,peripheral neuropathy, liver steatosis, osteoporosis with bone fracturesand avascular necrosis of the hips, and derangement of the complementsystem with coagulation disorders, leading to increased bleeding inhemophiliacs.

Regarding excess adipose tissue accumulation in HADDS, the keypathological abnormality associated with this syndrome, it is worthnoting that abnormal adipose accumulation is typically more pronouncedregionally than in the body as a whole (Carr et al, 1998a, 1998b;Engelson et al, 1999; Kotler et al, 1999). Since regional adiposedeposition in patients with HADDS may occur in conjunction withdepletion of subcutaneous adipose tissue elsewhere, and/or depletionlean body mass, whole body fat accumulation and weight may not changesignificantly as a patient develops HADDS. It should also be noted thatin order to diagnose HADDS, techniques that enable one to assessregional accumulation of adipose tissue are needed. Clinical assessmentmay include patient and provider history of changes in body habitus,anthropometry, serial photography, DXA, CT, and/or MRI scanning).

The most common presentation of regional fat accumulation in patientswith HADDS is truncal obesity or visceral adiposity. Carr et al (1998)documented increased truncal fat in a majority (64%) of their proteaseinhibitor-treated subjects using the technique of dual-energy X-rayabsorptiometry (DXA). Muurahainen at al (1999) found that 70 to 90%their cases reported increased abdominal girth as documented by patientself assessment questionnaire that was corroborated by physician report.

It has been demonstrated in that the truncal adipose tissue observed byDXA and increased abdominal girth reported by patients with HADDSprimarily reflects abnormal accumulation of visceral adipose tissue(Miller et al, 1998; Kotler et al, 1999; Engelson et al, 1999). Milleret al (1998) reported that some patients experienced an increase inabdominal girth with symptoms of abdominal fullness, distension, orbloating after adding indinavir, a protease inhibitor, to combinationdrug regimens for HIV-1 infection. In several patients with thesesymptoms, abdominal computed-tomography (CT) scans revealed an excessamount of intra-abdominal fat and a relative paucity of subcutaneousfat.

Using whole body magnetic imaging resonance (MRI) technology to quantifyabdominal fat, Kotler and colleagues reported that the increased girthin HIV patients with truncal obesity represented pathologicalaccumulation of visceral adipose tissue in conjunction with depletion ofsubcutaneous adipose tissue (Kotler et al, 1998; Engelson et al, 1999).Not all of these patient received HIV protease inhibitors. The visceraladipose tissue depot included omental adipose tissue surrounding theintestines along with pelvic and perinephric adipose tissue (Engelson etal, 1999). The visceral adipose tissue (VAT) accumulation observed inpatients with HADDS is typically two to seven times standard deviationsabove the mean for VAT in gender and age-matched healthy controls(Kotler, personal communication, 1999, manuscript in preparation).

Other types of abnormal adipose tissue accumulation in patients withHADDS include buffalo hump (dorsocervical fat pad), enlargement of thesubmandibular or supraclavicular fat pads with apparent bulging, lipomas(single or multiple) and increased accumulation of adipose tissue in thechest or breast area, observed more frequently in women more than in men(Dong K et al, 1998; Falutz, 1999; Gervasconi et al, 1999; Muurahainen,1999).

Regarding buffalo hump, Lo et al (1998) reported the results of studiesdone in eight HIV-1-positive patients referred for investigationabnormal accumulation of adipose tissue in the dorsocervical area. Only50% of these patients were receiving triple antiretroviral regimens thatincluded a protease inhibitor. No other signs of Cushing's Syndrome werepresent in the patients. The fact that 50% of the patients with buffalohump had no history of protease-inhibitor use indicates that developmentof non-Cushingoid buffalo hump is not unique to patients receivingprotease-inhibitor therapy. The mechanism of further increase intriglyceride values in patients with buffalo hump is not certain,although a possible relation between increased triglycerideconcentration and atypical body-fat distribution should be considered.For example, central fat accumulation may lead to the metabolic syndromeof insulin resistance, hypertriglyceridemia and hypertension if themajor component gained is visceral fat.

The subcutaneous adipose tissue depletion (lipodystrophy, orlipoatrophy) that is often observed HADDS patients includes thinning ofthe skin on the arms and legs, with venous prominence. There is alsothinning of facial fat with increased wrinkling of the face, especiallyin the nasolabial folds. Sometimes hollow sunken cheeks are observed.Viraben et al (1998) reported a case series involving eight patients whodeveloped either partial or generalized lipodystrophy after proteaseinhibitor therapy. While two patients developed progressive loss ofsubcutaneous fat from both legs, excess fat deposition in the unaffectedbuttocks and abdomen gave an impression of obesity. In six cases, acachetic appearance was observed resulting from the loss of buccal,parotid and preauricular fat pads. Two patients exhibited a generalizedloss of fatty tissue from the face. In 50% (four) of the patients inthis case series, either diabetes or insulin resistance was alsodiscovered.

Metabolic abnormalities do not occur in every patient with HADDS, andnot every HIV patient who develops metabolic abnormalities (whilereceiving protease inhibitors, other antiretroviral agents, or receivingno agents at all) concurrently develops abnormal fat accumulation.Mulligan et al (1998) compared results obtained in patients before andafter beginning an antiretroviral regimen that included a proteaseinhibitor or lamivudine. No significant changes in total or regional fator lean body mass were found by dual-energy X-ray absorptiometry in anygroup over the short time period of about four months. Miller et al(1998) found no relationships among CT-diagnosed visceral fataccumulation, hypertriglyceridemia, and hypercholesterolemia. Otherinvestigators have also reported that abnormal adipose tissueaccumulation in HIV patients is not always associated withhyperlipidemia or abnormal insulin/glucose metabolism (Lo et al, 1998;Muurahainen, 1999; Kotler and Schambelan; 1999).

Regarding disturbances of glucose and insulin metabolism, the FDAreported that there were reports of insulin resistance in patientsreceiving protease inhibitors for treatment HIV infection, and some ofthese diabetic patients were found to have truncal obesity (FDA, 1997).However, those who have since reviewed diabetes associated with the useof protease inhibitors have concluded that protease inhibitor-inducedhyperglycemia appears to be a rare occurrence (Dong B J et al, 1998;Keruly, 1998).

With respect to fasting hypertriglyceridemia, this abnormality was firstreported and associated with HIV infection, in the absence ofantiretroviral therapies and abnormal adipose tissue accumulation(Grunfeld, 1992). Patients with hypertriglyceridemia prior to startingcombination antiretroviral therapy appear to have an exacerbation of thecondition while on combination therapy and do not return to theirprevious state.

With respect to hypercholesterolemia and other metabolic abnormalitiesin HIV patients on combination antiretroviral therapy, serum cholesterolconcentrations rise, though rarely to dangerously high levels (Carr etal, 1998a, 1998b, 1999a, 1999b; Henry et al, 1998). Some patientsdeveloped hypertension and, in others, low serum testosteroneconcentrations developed or pre-existing hypogonadism persisted.

From the lack of tight associations described above, it is unclear ifadministration of protease inhibitors produces lipodystrophy syndrome orHADDS. Carr et al (1998), who described peripheral lipodystrophy andunusual fat accumulation in HIV-infected patients receiving proteaseinhibitors which, together with the known side effects of proteaseinhibitors of hyperlipidemia and diabetes mellitus, have suggested thatprotease inhibitors cause metabolic perturbations leading to insulinresistance. Carr also found evidence of insulin resistance in patientswith lipodystrophy syndrome, although clinically apparent diabetesmellitus was very uncommon. An amino acid sequence in the catalytic siteof HIV protease was found to have a significant homology with a lowdensity lipoprotein receptor-like protein. These results tend toimplicate the protease inhibitors themselves in the development of thisproblem.

However, other researchers have found that the protease inhibitors perse are not always associated with the morphologic and metabolicabnormalities of lipodystrophy syndrome and HADDS (Lo et al, 1998;Kotler et al, 1998; Brinkman 1999; Carr, 1999b; Gervasconi, 1999;Hadigan et al, 1999). Morphologic and metabolic changes are not seenconsistently in all protease inhibitor-treated patients, and manypatients have only certain elements of the syndrome. In some cases theabnormal morphologic and/or metabolic manifestations have been observedin patients who have never received any type of antiretroviralmedication (Lo et al, 1998; Carr et al, 1998a, 1998b, 1999a, 1999b;Mercie et al, 1999).

Even though HIV patients with visceral adiposity and buffalo hump havephenotypes that are reminiscent of Cushing's syndrome, it is unlikelythat HADDS patients will have Cushing's syndrome. In a case series ofHIV patients with buffalo hump, Lo et al (1998) found that fasting serumcortisol concentrations and standard dexamethasone suppression testswere normal. In a case series of HIV patients with abnormal visceraladipose tissue accumulation (Kotler et al, 1998), and in one of thepatients of with buffalo hump (Lo et al, 1998) moderate elevations in24-hour urinary-free cortisol were observed. However, even though theremay be subtle abnormalities in cortisol in patients with HADDS, so farCushing's syndrome has been ruled out in all of them by the standarddexamethasone suppression test.

As mentioned above, the possibility of concomitant AIDS wasting and/ordepletion of lean body mass a consideration in HADDS. Despite advancesin treating the retrovirus infection and the complications of AIDS, someHIV-infected patients still develop overt AIDS wasting (or cachexia),which involves profound depletion of weight and lean body mass. OvertAIDS wasting, defined as an involuntary weight loss exceeding 10% ofusual body weight, has become less prevalent since the widespread use ofhighly active antiretroviral therapy, and probably occurs in fewer than10% of patients receiving highly active antiretroviral therapy (Mocroft,1999). Similarly, in one large case series, fewer than 10% of HADDSpatients exhibited overt AIDS wasting (Muurahainen et al, unpublishedobservations).

However, HIV patients may also present with “occult wasting”, defined asa significant depletion of lean body mass without significant weightloss (Muurahainen, 1994). The prevalence of occult wasting in patientswith HADDS is currently under investigation. Since occult wasting mayoccur in patients receiving combination antiretroviral therapy(Muurahainen, 1994; Gibert, 1996), it is possible that patients withHADDS will also display depletion of lean body mass without weight loss,or occult wasting.

To summarize, HADDS is newly discovered a HIV-related syndrome involvingrelatively rapid, pathologic accumulation of adipose tissue in specificdepots that develops over a period of several months to years,frequently in association with subcutaneous peripheral lipodystrophy(facial and limb wasting), and occasionally associated withhyperlipidemia and hyperinsulinemia, in the absence of hypercortisolism.The syndrome may also be associated with other physiological changessuch as premature atherosclerosis, depletion of lean body mass, andother physiologic abnormities. As such, HADDS is a unique clinicalentity in the history of medicine.

With respect to the invention described herein, it should be noted thata small percentage (probably fewer than ten percent) of HADDS patientsin the United States have overt AIDS wasting (Muurahainen et al,unpublished observations, Serono Laboratories, Inc), although inprobably more, if not all HADDS patients, occult wasting may be present.It should be noted that many patients with overt AIDS wasting are ableto tolerate relatively high doses (6 mg/day) of recombinant growthhormone (rhGH; Serono's SEROTSIM) administered subcutaneously (s.c.),without developing adverse effects that require dose reduction orcessation of therapy (Schambelan et al, 1996).

In comparison to AIDS wasting patients, healthy (non-HIV infected,non-wasting) adults, when given 6 mg/day s.c. injections of rhGH willprobably develop more symptomatology such as tissue turgor, jointstiffness, arthalgias, and/or paresthesias necessitating dose reductionor cessation of therapy (data on file, Serono Laboratories, Inc/,Norwell, Mass.). Dose-ranging trials are currently being designed toinvestigate the most effective and safe doses of rhGH for patients withHADDS, and to ascertain whether HADDS patients with occult and overtwasting are able to tolerate higher doses than HADDS patients withoutovert or occult wasting, if any.

Also with respect to the invention proposed herein, it should be notedthat Bjorntorp and others (1996) have reported a disease entity termedmetabolic syndrome X (or syndrome X), occurring in non-HIV-infectedadults, that bears several similarities to HADDS. Patients with syndromeX typically present with mild to moderate visceral truncal adiposity,hyperlipidemia, insulin resistance, sometimes diabetes and hypertension,and occasionally associated with signs of premature atherosclerosis.However, this non-HIV related syndrome typically develops over manyyears. Moreover, compared to HADDS, syndrome X is not typicallyassociated with (1) chronic HIV infection (2) excessive accumulation ofadipose tissue in the visceral, dorsocervical, submandibular,supraclavicular, pectoral and/or mammary areas, (3) occasionallyprofound lipodystrophy or lipoatrophy of subcutaneous fat in the face,arms, legs, and/or buttocks, (4) lipomas, either multiple or single (ifany at all), and (5) wasting, either overt or occult.

Other than for the invention proposed below, there is no currently knownmedical therapy that effectively treats the primary pathologicalabnormality of HADDS. Of course, there are a few approaches to thetreatment of several abnormalities occasionally observed in patientswith HADDS, such as hyperlipidemia, hyperinsulinemia/diabetes,wasting/cachexia, pancreatitis, gout, and hypogonadism. Sometimes theseconditions can be managed in HADDS patients almost exactly as managed innon-HIV-infected patients.

However, the potential for reduction of the abnormal adipose tissueaccumulation is uncertain. Since visceral adiposity, buffalo hump, andother fat accumulation in HIV-1-infected individuals with HADDS does notthe result from Cushing's Syndrome (Lo et al, 1998), it would be unwiseto treat it as such. Dietary modification and exercise are of limitedsuccess in reducing abnormally distributed regional fat accumulationsuch as buffalo hump and visceral adiposity (Kotler et al, personalcommunication). As described above, antiretroviral switch strategieshave been disappointing at best. Saint-Marc (1999) has reported that theantihyperglycemic agent metformin may result in reduced total,subcutaneous, and visceral fat. However, because of its adverse effectprofile, metformin may not be the best therapeutic agent to offer tomost patients with HADDS.

Surgical interventions, such as liposuction and surgical excision, havebeen used with limited success to treat abnormally accumulated adiposetissue in the dorsocervical, submandibular, breast areas, or as lipomas.It is not feasible to liposuction abnormally accumulated visceraladipose tissue due to the risk of bowel perforation. Liposuction orplastic surgery produce only temporary relief, because the abnormal fattypical re-accumulates following surgical excision. Repeat surgicalprocedures augment risks of anesthesia and scarring. Moreover, thesurgical techniques do not affect the metabolic abnormalities sometimesassociated with HADDS.

SEROSTIM®, recombinant human growth hormone (rhGH) produced by SeronoLaboratories, Inc., has recently been given accelerated FDA approval fortreating wasting syndrome in patients with AIDS or AIDS-relatedcachexia.

Windisch et al (1998) reported that AIDS-associated wasting wascharacterized by weight loss, depletion of lean body mass andpreservation of body fat, leading to muscle weakness and organ failure.Although the FDA has approved recombinant growth hormone for treatingAIDS-associated wasting, the adverse event profile is similar to that ofother recombinant growth hormone products. Trials of recombinant growthhormone on the control of wasting in patients with AIDS have beenencouraging. Post-marketing experience with over 10,000 AIDS wastingpatients receiving SEROSTIM® since 1996 reveals that a three-monthcourse of therapy was effective in the majority of patients with AIDSwasting.

Krentz et al (1993) compared metabolic and anthropometric changesinduced by recombinant human growth hormone dosed at 5.0 versus 2.5 mgevery other day (qod) in 10 patients with HIV/AIDS. During treatment,insulin-like growth factor-1 (IGF-1) levels increased significantly inthe pharmacological rhGH treatment group receiving 5.0 mg qod, whereasno significant change was observed in IGF-1 in the group receiving 2.5mg qod of rhGH. In the group treated with 5.0 mg qod dose of hGH, weightloss preceding the study was reversed in each of the four patients whocompleted the study. This weight gain was associated with increases inlean body mass and total body water, and with concomitant decreases infat mass and urinary nitrogen excretion.

In a large, randomized, placebo-controlled study, Schambelan et al(1996) used dual X-ray absorptiometery (DXA) scanning to evaluatechanges in body composition produced by administration of recombinanthuman growth hormone dosed at 0.1 mg/kg/day (or 4 to 6 kg per day,depending on patient weight) compared to placebo over a 12 weeks courseof therapy. By the end of treatment, significant increases in lean bodymass and weight were observed in the rhGH group, compared to the placebogroup, and these increases correlated with improvements in physicalfunction (treadmill performance). The rhGH therapy was associated withminor increments in fasting plasma glucose, which were of negligibleclinical significance.

SUMMARY OF THE INVENTION

It is an object of the present invention to overcome the aforementioneddeficiencies in the prior art.

It is another object of the present invention to treat any type of HADDSand related syndromes, including excess accumulation of adipose tissuein the visceral, submandibular, supraclavicular, dorsocervical,pectoral, and/or mammary areas, as well as subcutaneous lipomas (whichmay occur singly or multiply), with recombinant human growth hormone(rhGH) or any other substance which binds to and initiates signalling ofthe human growth hormone receptor or which stimulates release of orpotentiates the activity of endogenous hGH.

According to the present invention, human growth hormone (hGH) isadministered to treat HADDS and possibly related syndromes, such asmetabolic syndrome X (or syndrome X) in patients without HIV infection,or any non-HIV related lipodystrophy syndrome (hereditary or acquired)that includes abnormal adipose tissue accumulation.

Because HIV patients are more likely to have wasting thannon-HIV-infected patients, and because patients with wasting appearbetter able to tolerate supraphysiologic doses of rhGH than non-wastingpatients, it is postulated that, if supraphysiologic doses of rhGH arerequired to reduce abnormally accumulated adipose tissue, the therapywill be better tolerated by patients with HADDS than non-HIV-infectedpatients with related syndromes. Nevertheless, in one embodiment of thepresent invention, the treatment is only directed to HADDS patients whodo not present with AIDS wasting.

The human growth hormone administered is preferably recombinant humangrowth hormone (rhGH). Alternatively, a substance which stimulatesrelease of endogenous growth hormone, such as growth hormone releasinghormone (GHRH) or other substances which agonize the GHRH receptor, maybe used. Any HADDS patient can be treated by means of the presentinvention.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention relates to the discovery that HIV-associateddysmorphia/dysmetabolic syndrome (HADDS) may be treated by theadministration of an effective amount of human growth hormone. The HADDSwhich may be treated in accordance with the present invention maypresent itself as, for example, dorsocervical fat pads (“buffalo hump”),visceral adiposity (“truncal obesity”, “omental fat accumulation”, “Crixbelly” or “protease paunch”), abnormal pectoral or mammary fataccumulation, and other abnormal fat accumulation in other depots(including submandibular fat accumulation or “horse collar”, andlipomas, either single, multiple, or bilateral symmetric lipomatoses).

Human growth hormone, also known as somatotropin, is a protein hormoneproduced and secreted by the somatotropic cells of the anteriorpituitary. Secretion is regulated by a releasing factor, i.e., thegrowth hormone-releasing hormone (GHRH), and by an inhibitory factor,somatostatin. Human growth hormone plays a key role in somatic growththrough its effects on the metabolism of proteins, carbohydrates andlipids.

Human growth hormone is a single polypeptide chain of 191 amino acids(Bewley et al, 1972) having two disulfide bonds, one between Cys-53 andCys-165, forming a large loop in the molecule, and the other betweenCys-182 and Cys-189, forming a small loop near the C-terminus. The DNAsequence that confirmed the amino acid sequence was reported by Martialet al (1979). Purified hGH is a white amorphous powder in itslyophilized form. It is readily soluble (concentrations >10 mg/L) indilute aqueous buffers at pH greater than 7.2.

In solution, hGH exists predominantly as a monomer, with a smallfraction as dimers and higher molecular weight oligomers. Under certainconditions, hGH can be induced to form larger amounts of dimers, trimersand higher oligomers.

Several derivatives of hGH are known, including naturally-occurringderivatives, variants and metabolic products, degradation productsprimarily of biosynthetic hGH and engineered derivatives of hGH producedby genetic methods. One example of a naturally-occurring derivative ofhGH is GH-V, a variant of growth hormone found in the placenta. Othermembers of the gene locus are described in Chen et al (1989). Anyderivative of hGH, including derivatives designed to be long-lasting inthe body, can be used for the purpose of the present invention as longas it retains the biological activity of hGH.

Methionyl hGH was the first form of hGH to be produced throughrecombinant DNA technology. This compound is actually a derivative ofhGH having one additional methionine residue at its N-terminus (Goeddelet al, 1979).

A naturally-occurring variant of hGH called 20-K-hGH has been reportedto occur in the pituitary as well as in the bloodstream (Lewis et al,1978; Lewis et al, 1980). This compound, which lacks the 15 amino acidresidues from Glu-32 to Gln-46, arises from an alternative splicing ofthe messenger ribonucleic acid (DeNoto et al, 1981). This compoundshares many, but not all of the biological properties of hGH.

20-K-HGH is made in the pituitary and secreted into the blood. It makesup about 5% of growth hormone output of adults, and about 20% of growthhormone output of children. It has the same growth promoting activity as22 kD growth hormone, and has been reported to have equal to or greaterthe amount of lipolytic activity as the 22 kD form. It binds to growthhormone receptors with equal affinity as the 22 kD growth hormone, andhas one tenth the lactogenic (prolactin-like) bioactivity as the 22 kDhormone. Unlike 22 kD, the 2-k-HGH has weak anti-insulin activity.

A number of derivatives of hGH arise from proteolytic modifications ofthe molecule. The primary pathway for the metabolism of hGH involvesproteolysis. The region of hGH around residues 130-150 is extremelysusceptible to proteolysis, and several derivatives of hGH having nicksor deletions in this region have been described (Thorlacius-Ussing,1987). This region is in the large loop of hGH, and cleavage of apeptide bond there results in the generation of two chains that areconnected through the disulfide bond at Cys-53 and Cys-165. Many ofthese two-chain forms are reported to have increased biological activity(Singh et al, 1974). Many derivatives of human growth hormone have beengenerated artificially through the use of enzymes. The enzymes trypsinand subtilisin, as well as others, have been used to modify hGH atvarious points throughout the molecule (Lewis et al, 1977b; Graff et al,1982). One such derivative, called two-chain anabolic protein (2-CAP),was formed through the controlled proteolysis of hGH using trypsin(Becker et al, 1989). 2-CAP was found to have biological properties verydistinct from those of the intact hGH molecule, in that thegrowth-promoting activity of hGH was largely retained and most of theeffects on carbohydrate metabolism were abolished.

Asparagine and glutamine residues in proteins are susceptible todeamidation reactions under appropriate conditions. Pituitary hGH hasbeen shown to undergo this type of reaction, resulting in conversion ofAsn-152 to aspartic acid and also, to a lesser extent, conversion ofGln-137 to glutamic acid (Lewis et al, 1981). Deamidated hGH has beenshown to have an altered susceptibility to proteolysis with the enzymesubtilisin, suggesting that deamidation may have physiologicalsignificance in directing proteolytic cleavage of hGH. Biosynthetic hGHis known to degrade under certain storage conditions, resulting indeamidation at a different asparagine (Asn-149). This is the primarysite of deamidation, but deamidation at Asn-152 is also seen (Becker etal, 1988). Deamidation at Gln-137 has not been reported in biosynthetichGH.

Methionine residues in proteins are susceptible to oxidation, primarilyto the sulfoxide. Both pituitary-derived and biosynthetic hGH undergosulfoxidations at Met-14 and Met-125 (Becker et al, 1988). Oxidation atMet-170 has also been reported in pituitary but not biosynthetic hGH.Both desamide hGH and Met14 sulfoxide hGH have been found to exhibitfull biological activity (Becker et al, 1988).

Truncated forms of hGH have been produced, either through the actions ofenzymes or by genetic methods. 2-CAP, generated by the controlledactions of trypsin, has the first eight residues at the N-terminus ofhGH removed. Other truncated versions of hGH have been produced bymodifying the gene prior to expression in a suitable host. The first 13residues have been removed to yield a derivative having distinctivebiological properties (Gertler et al, 1986) in which the polypeptidechain is not cleaved.

Although human growth hormone was originally obtained from pituitaryglands of cadavers, these preparations were not electorphoreticallyhomogeneous, and antibodies appeared in the serum of patients treatedwith preparations of the order of 50% purity, the immunogenicity beingattributed to inactive components. Recombinant DNA technology permittedproduction of an unlimited supply of hGH in a number of differentsystems. Purification of hGH from the culture medium is facilitated bythe presence of only low amounts of contaminating proteins. In fact, ithas been shown that hGH can be purified on a laboratory scale by asingle purification step on a reversed-phase HPLC column (Hsiung et al,1989).

Recombinant human growth hormone, rhGH, is produced by SeronoLaboratories, Inc., as SEROSTIM®, which product has been givenaccelerated FDA approval for treating weight loss and wasting in AIDSpatients. PROTROPIN®, produced by Genentech, Inc. (South San Francisco,Calif.), differs slightly in structure from natural sequence hGH, havingan additional methionine residue at the N-terminus. Recombinant hGH isgenerally marketed as vials containing hGH plus additional excipients,e.g., glycine and mannitol, in a lyophilized form. A companion diluentvial is provided, allowing the patient to reconstitute the product tothe desired concentration prior to administration of the dose.Recombinant hGH can also be marketed in other well-known manners, suchas prefilled syringes, etc.

After intravenous administration, the elimination of hGH is described byfirst-order kinetics with a serum half-life of 1230 minutes in bothanimals and humans (Moore et al, 1988; Hendricks et al, 1985).Traditionally, intramuscular injection has been the method of choice asthe preferred route of delivery. In humans, absorption of exogenous hGHappears to be more rapid from the intramuscular site, with a time tomaximum concentration of two to three hours, compared to four to sixhours after subcutaneous administration. The disappearance phase fromserum has been reported to range from 12-20 hours for intramuscularadministration, and 20-24 hours after subcutaneous administration(Albertsson-Wikland et al, 1986; Jorgensen et al, 1987). In general, nosignificant differences have been observed in the pharmacokinetics orbiological activities of recombinant natural sequence hGH, recombinantN-methionyl-hGH, or pituitary-derived material in humans (Moore et al,1988; Jorgensen et al, 1988).

The term “human growth hormone”, as used in the present invention, isintended to include the naturally-occurring derivatives, as noted above,including, without limitation, both the 20 kD and the 22 kD human growthhormone, GH-V, and other members of the growth hormone gene locus asdescribed in Chen et al (1989). The term also includes functionalderivatives, fragments, variants, analogs, or salts which retain thebiological activity of growth hormone, i.e., which act as agonists tothe growth hormone receptor. In other words, they are capable of bindingto the growth hormone receptor to initiate the signaling activity of thereceptor.

“Functional derivatives” as used herein covers derivatives which may beprepared from the functional groups which occur as side chains on theresidues or the N- or C-terminal groups, by means known in the art, andare included in the invention as long as they remain pharmaceuticallyacceptable, i.e., they do not destroy the biological activity of hGH asdescribed herein, i.e., the ability to bind the hGH receptor andinitiate receptor signalling, and do not confer toxic properties oncompositions containing it. Derivatives may have chemical moieties, suchas carbohydrate or phosphate residues, provided such a derivativeretains the biological activity of hGH and remains pharmaceuticallyacceptable.

For example, derivatives may include aliphatic esters of the carboxylgroups, amides of the carboxyl groups by reaction with ammonia or withprimary or secondary amines, N-acyl derivatives or free amino groups ofthe amino acid residues formed with acyl moieties (e.g., alkanoyl orcarbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl group(e.g., that of seryl or threonyl residues) formed with acyl moieties.Such derivatives may also include for example, polyethylene glycolside-chains which may mask antigenic sites and extend the residence ofthe molecule in body fluids.

Of particular importance is a growth hormone that has been derivatizedor combined with a completing agent to be long lasting. For example,pegylated versions, or growth hormones genetically engineered to exhibitlong lasting activity in the body, can be used to treat HADDS accordingto the present invention.

HGH that is acetylated at the N-terminus has been isolated andidentified (Lewis et al, 1979). It is not clear if acylation serves aregulatory role or is simply an artifact of the purification. However,it is expected that this the molecule exhibits anti-HADDS activity in asimilar fashion to other hGH derivatives.

The term “derivatives” is intended to include only those derivativesthat do not change one amino acid to another of the twentycommonly-occurring natural amino acids.

The term “salts” herein refers to both salts of carboxyl groups and toacid addition salts of amino groups of the hGH molecule or analogsthereof. Salts of a carboxyl group may be formed by means known in theart and include inorganic salts, for example, sodium, calcium, ammonium,ferric or zinc salts, and the like, and salts with organic bases asthose formed, for example, with amines, such as triethanolamine,arginine or lysine, piperidine, procaine and the like. Acid additionsalts include, for example, salts with mineral acids, such as, forexample, hydrochloric acid or sulfuric acid, and salts with organicacids, such as, for example, acetic acid or oxalic acid. Of course, anysuch salts must retain the biological activity of hGH relevant to thepresent invention, i.e., the ability to bind to the hGH receptor andinitiate receptor signalling.

A “fragment” of the growth hormone according to the present inventionrefers to any subset of the molecule, that is, a shorter peptide whichretains the desired biological activity. Fragments may readily beprepared by removing amino acids from either end of the hGH molecule andtesting the resultant for its properties as an hGH receptor agonist.Proteases for removing one amino acid at a time from either theN-terminal or the C-terminal of a polypeptide are known, and sodetermining fragments which retain the desired biological activityinvolves only routine experimentation.

Additionally, the polypeptide which has such hGH receptor agonistactivity, be it hGH, an analog or variant, salt, functional derivativeor fragment thereof, can also contain additional amino acid residuesflanking the hGH polypeptide. As long as the resultant molecule retainsthe hGH receptor agonist ability of the core polypeptide, one candetermine whether any such flanking residues affect the basic and novelcharacteristics of the core peptide, i.e., its receptor agonistcharacteristics, by routine experimentation. The term “consistingessentially of”, when referring to a specified sequence, means thatadditional flanking residues can be present which do not affect thebasic and novel characteristic of the specified sequence. This term doesnot comprehend substitutions, deletions or additions within thespecified sequence.

A “variant” of the human growth hormone according to the presentinvention refers to a molecule which is substantially similar to eitherthe entire peptide or a fragment thereof. Variant peptides may beconveniently prepared by direct chemical synthesis of the variantpeptide, using methods well known in the art. Of course, a variant humangrowth hormone would have similar similar hGH receptor binding andsignal initiating activity as hGH and which would, therefore, beexpected to have similar anti-HADDS activity to hGH.

Amino acid sequence variants of the human growth hormone can be preparedby mutations in the DNAs which encode the synthesized human growthhormone derivatives. Such variants include, for example, deletions from,or insertions or substitutions of, residues within the amino acidsequence. Any combination of deletion, insertion, and substitution mayalso be made to arrive at the final construct, provided that the finalconstruct possesses the desired activity. Obviously, the mutations thatwill be made in the DNA encoding the variant peptide must not alter thereading frame and preferably will not create complementary regions thatcould produce secondary mRNA structure (cf. European Patent PublicationNo. EP 75,444, the entire contents of which being hereby incorporated byreference).

At the genetic level, these variants ordinarily are prepared bysite-directed autogenesis (as exemplified by Adelman et al, 1983) ofnucleotides in the DNA encoding the peptide molecule, thereby producingDNA encoding the variant, and thereafter expressing the DNA inrecombinant cell culture. The variants typically exhibit the samequalitative biological activity as the non-variant peptide.

An “analog” of human growth hormone according to the present inventionrefers to a non-natural molecule which is substantially similar toeither the entire molecule or to an active fragment thereof. An analogof human growth hormone useful in the present invention would exhibitanti-HADDS activity.

The types of substitutions which may be made in the human growth hormoneaccording to the present invention may be based on analysis of thefrequencies of amino acid changes between a homologous protein ofdifferent species. Based upon such analysis, conservative substitutionsmay be defined herein as exchanges within one of the following fivegroups:

I. Small, aliphatic, nonpolar or slightly polar residues:

Ala, Ser, Thr, Pro, Gly

II. Polar, negatively-charged residues and their amides:

Asp, Asn, Glu, Gln

III. Polar, positively-charged residues:

His, Arg, Lys

IV. Large, aliphatic non-polar residues:

Met, Leu, Ile, Val, Cys

V. Large aromatic residues:

Phe, Try, Trp

Within the foregoing groups, the following substitutions are consideredto be “highly conservative”:

Asp/Glu

His/Arg/Lys

Phe/Tyr/Trp

Met/Leu/Ile/Val

Semi-conservative substitutions are defined to be exchanges between twoof groups (I)-(IV) above which are limited to supergroup (A), comprising(I), (II), and (III) above, or to supergroup (B), comprising (IV) and(V) above. Substitutions are not limited to the genetically encoded oreven the naturally occurring amino acids. When the epitope is preparedby peptide synthesis, the desired amino acid may be used directly.Alternatively, a genetically encoded amino acid may be modified byreacting it with an organic derivatizing agent that is capable ofreacting with selected side chains or terminal residues.

Cysteinyl residues most commonly are reacted with alpha-haloacetates(and corresponding amines), such as chloroacetic acid orchloroacetamide, to give carboxylmethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, alpha-bromo-beta-(5imidazoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl-2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Parabromophenacyl bromide is also useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino acid-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;0-methyliosurea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal; 2,3butanedione; andninhydrin. Derivatization of arginine residues requires that thereaction be performed in alkaline conditions because of the high pKa ofthe guanidine functional group. Furthermore, these reagents may reactwith the groups of lysine, as well as the arginine epsilon-amino group.

The specific modification of tyrosyl residues per se has been studiedextensively, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidazole andtetranitromethane are used to form O-acetyl tyrosyl species and e-nitroderivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′N—C—N—R′), such as1-cyclohexyl-3-[2-morpholinyl-(4-ethyl)]carbodiimide or 1ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyland glutamyl residues are converted to asparaginyl and glutaminylresidues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Examples of production of amino acid substitutions in proteins which canbe used for obtaining analogs of the hGH for use in the presentinvention include any known method steps, such as presented in U.S. Pat.Nos. RE 33,653; 4,959,314; 4,588,585 and 4,737,462, to Mark et al; U.S.Pat. No. 5,116,943 to Koths et al; U.S. Pat. No. 4,965,195 to Namen etal; and U.S. Pat. No. 5,017,691 to Lee, et al, and lysine substitutedproteins presented in U.S. Pat. No. 4,904,584 (Shaw et al).

Among the substances which bind to and initiate signalling of the humangrowth hormone receptor which may be used in accordance with the presentinvention are all of those growth hormone analogs and mimetics alreadyknown in the literature, such as, for example, are disclosed in U.S.Pat. Nos. 5,851,992; 5,849,704; 5,849,700; 5,849,535; 5,843,453;5,834,598; 5,688,666; 5,654,010; 5,635,604; 5,633,352; 5,597,709; and5,534,617.

Preferably, the hGH variant or analog will have a core sequence, whichis the same as that of the native sequence or biologically activefragment thereof, which has an amino acid sequence having at least 70%identity to the native amino acid sequence and retains the biologicalactivity thereof. More preferably, such a sequence has at least 80%identity, at least 90% identity, or most preferably at least 95%identity to the native sequence.

The term “sequence identity” as used herein means that the sequences arecompared as follows. The sequences are aligned using Version 9 of theGenetic Computing Group's GAP (global alignment program), using thedefault (BLOSUM62) matrix (values −4 to +11) with a gap open penalty of−12 (for the first null of a gap) and a gap extension penalty of −4 (pereach additional consecutive null in the gap). After alignment,percentage identity is calculated by expressing the number of matches asa percentage of the number of amino acids in the claimed sequence.

Analogs or variants in accordance with the present invention may also bedetermined in accordance with the following procedure. The DNA of thenative sequence is known to the prior art and is found in the literature(Martial et al, 1979). Polypeptides encoded by any nucleic acid, such asDNA or RNA, which hybridizes to the complement of the native DNA or RNAunder highly stringent or moderately stringent conditions, as long asthat polypeptide maintains the biological activity of the nativesequence, are also considered to be within the scope of the presentinvention.

Stringency conditions are a function of the temperature used in thehybridization experiment, the molarity of the monovalent cations and thepercentage of formamide in the hybridization solution. To determine thedegree of stringency involved with any given set of conditions, onefirst uses the equation of Meinkoth et al. (1984) for determining thestability of hybrids of 100% identity expressed as melting temperatureTm of the DNA-DNA hybrid:

Tm=81.5° C.+16.6(Log M)+0.41(% GC)−0.61(% form)−500/L

where M is the molarity of monovalent cations, % GC is the percentage ofG and C nucleotides in the DNA, % form is the percentage of formamide inthe hybridization solution, and L is the length of the hybrid in basepairs. For each 1° C. that the Tm is reduced from that calculated for a100% identity hybrid, the amount of mismatch permitted is increased byabout 1%. Thus, if the Tm used for any given hybridization experiment atthe specified salt and formamide concentrations is 10° C. below the Tmcalculated for a 100% hybrid according to equation of Meinkoth,hybridization will occur even if there is up to about 10% mismatch.

As used herein, highly stringent conditions are those which are tolerantof up to about 15% sequence divergence, while moderately stringentconditions are those which are tolerant of up to about 20% sequencedivergence. Without limitation, examples of highly stringent (12-15° C.below the calculated Tm of the hybrid) and moderately (15-20° C. belowthe calculated Tm of the hybrid) conditions use a wash solution of 2×SSC(standard saline citrate) and 0.5% SDS at the appropriate temperaturebelow the calculated Tm of the hybrid. The ultimate stringency of theconditions is primarily due to the washing conditions, particularly ifthe hybridization conditions used are those which allow less stablehybrids to form along with stable hybrids. The wash conditions at higherstringency then remove the less stable hybrids. A common hybridizationcondition that can be used with the highly stringent to moderatelystringent wash conditions described above is hybridization in a solutionof 6×SSC (or 6×SSPE), 5×Denhardt's reagent, 0.5% SDS, 100 μg/mldenatured, fragmented salmon sperm DNA at a temperature approximately20° to 25° C. below the Tm. If mixed probes are used, it is preferableto use tetramethyl ammonium chloride (TMAC) instead of SSC (Ausubel,1987-1998).

While the present invention provides recombinant methods for making thehuman growth hormone derivatives, these derivatives may also be made byconventional protein synthesis methods which are well known to thoseskilled in the art.

The growth hormone treatment in accordance with the present inventionmay be accomplished either by administration of exogenous growth hormoneor by administration of a substance which stimulates production ofendogenous growth hormone either directly or indirectly by supressingendogenous somatostatin secretion. It is known that human growth hormonereleasing hormone (hGHRH) stimulates the release of hGH. Thus, thebiological activity of hGH can be indirectly obtained by administeringGHRH or a functional derivative, salt, variant, analog or fragmentthereof which retains the biological activity of GHRH, i.e., the abilityto stimulate the release of growth hormone. Thus, for example, besidesGHRH there may be used functional derivatives thereof in accordance withthe above definition, analogs or variants thereof, which have at least70% sequence identity, more preferably 80% or 90% or, most preferably,95% sequence identity therewith, yet retains the biological activity ofGHRH, or a variant or analog which is a polypeptide encoded by a DNAwhich hybridizes to the native DNA encoding GHRH under moderatelystringent conditions, or preferably under highly stringent conditions,all in accordance with the definitions given hereinabove. Any of theGHRH or GHRH analogs or agonists known in the literature and disclosedas simulating the release of growth hormone can be used in the presentinvention, such as those disclosed in U.S. Pat. Nos. 5,792,747;5,776,901; 5,696,089; 5,137,872; 5,767,085; 5,612,470; 5,846,936; and5,847,066. See also Thorner et al (1997), Felix et al (1995), Alba-Rothet al (1988), Friend et al (1997).

Other substances capable of promoting the release of growth hormone invivo which can be used in accordance with the present invention includethose disclosed in U.S. Pat. Nos. 5,807,985; 5,804,578; 5,795,957;5,777,112; 5,767,118; 5,731,317; 5,726,319; 5,726,307; 5,721,251;5,721,250, etc.

There can also be used in accordance with the present invention anyother molecule which binds to the hGH receptor and initiates signallingof that receptor. It is known, for example, that small molecules,sometimes called secretagogues, have been developed which bind hGHreceptors and cause them to aggregate and initiate signalling, whichsignal initiation is the same as one obtains with natural hGH binding tothe receptor. Such molecules are known, for example, from U.S. Pat. Nos.5,773,441; 5,798,337; 5,830,433; 5,767,124; and 5,723,616. See alsoBowers et al (1991), Thorner et al (1997), Camanni et al (1998),Ankersen et al (1998), Smith et al (1993) and Ghigo et al (1998). Thus,the present invention is intended to include any substance which bindsto hGH receptor and initiates signalling thereof so as to obtain thesame ultimate qualitative effect as the administration of natural hGH,insofar as the treatment of HADDS is concerned.

Pharmaceutical compositions for administration according to the presentinvention can comprise at least one human growth hormone according tothe present invention in a pharmaceutically acceptable form optionallycombined with a pharmaceutically acceptable carrier. These compositionscan be administered by any means that achieve their intended purposes.Amounts and regimens for the administration of a composition accordingto the present invention can be determined readily by those withordinary skill in the art for treating HADDS.

For example, administration can be by parenteral, such as subcutaneous,intravenous, intramuscular, intraperitoneal, aerosol, or trahsdermalroutes. The dosage administered depends upon the age, health and weightof the recipient, type of previous or concurrent treatment, if any,frequency of the treatment and the nature of the effect desired.

Compositions within the scope of this invention include all compositionscomprising at least one human growth hormone or derivative, analog, orvariant thereof according to the present invention in an amounteffective to achieve its intended purpose. While individual needs vary,determination of optimal ranges of effective amounts of each componentis within the skill of the art. Typical dosages comprise about 0.01 toabout 0.1 mg/kg body weight per day, which will usually amount to about1-6 mg/day, subcutaneously for 5-30 weeks. When administered to AIDSpatients, the hGH anti-HADDS therapy may be administered concomitantlywith other AIDS therapies. Since supraphysiologic doses of hGH (>5mg/day) have been safely administered to AIDS wasting patientscontinuously on a daily basis as s.c. injections for periods of two tofour years (data on file, Serono Laboratories, Inc), in HADDS patientsin whom the abnormal adipose tissue re-accumulates, re-treatment ormaintenance therapies will be considered.

It should also be understood that, to be useful, the treatment providedneed not be absolute, provided that it is sufficient to carry clinicalvalue. An agent which provides treatment to a lesser degree than docompetitive agents may still be of value if the other agents areineffective for a particular individual, if it can be used incombination with other agents to enhance the overall level ofprotection, or if it is safer than competitive agents.

It is understood that the suitable dose of a composition according tothe present invention will depend upon the age, health and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired. However, the most preferred dosagecan be tailored to the individual subject, as is understood anddeterminable by one of skill in the art, without undue experimentation.This typically involves adjustment of a standard dose, e.g., reductionof the dose if the patient has a low body weight.

The total dose required for each treatment may be administered inmultiple doses or in a single dose. The compositions may be administeredalone or in conjunction with other therapeutics directed to the diseaseor directed to other symptoms thereof.

In addition to the compounds of the invention, a pharmaceuticalcomposition may contain suitable pharmaceutically acceptable carriers,such as excipients, carriers and/or auxiliaries which facilitateprocessing of the active compounds into preparations which can be usedpharmaceutically.

EXPERIMENTAL

Eight AIDS patients, six male and two female, with a history oflong-term use (an average of 12 months) of highly active antiretroviraltherapy (HAART), including protease inhibitors (6 indinavir, 2ritonavir/saquinavir) developed HADDS, including buffalo humps, centraladiposity and peripheral muscle wasting associated with fatigue, alongwith elevated levels of plasma triglycerides +/− cholesterol.

Therapy with rhGH (SEROSTIMw,w) was initiated in all patients at a doseof 6 mg/day subcutaneously. Four patients completed three months of rhGHand had notable improvements in fat maldistribution, with 25-75%reduction in buffalo hump syndrome and abdominal girth, but no change inperipheral lipodystrophy. Weights were stable, and there were noconsistent changes in total body fat and blood lipids, despite 5-10%gain in fat-free mass. One patient discontinued rhGH due to carpaltunnel syndrome and had recurrence of HADDS. Three patients have hadover six months of therapy. One patient was lost to follow-up after sixweeks of therapy, and one patient has received fewer than eight weeks oftherapy. Yet, at last observation, all had notable reductions in thesize of and firmness of the buffalo hump and truncal adiposity. Theseexperiments establish that rhGH is effective in treating HADDS,including reduction of buffalo humps and truncal adiposity.

Since the time that this invention was initially reported, thesefindings have been replicated in over 40 HIV patients with HADDS (Torreset al, 1998; Torres, 1999; Wanke et al, 1999; Mauss et al, 1999;Engleson et al 1999; Milano et al, 1999). Torres (1999) has reportedthat, by four months, rhGH therapy dosed at 4 to 6 mg/day significantlyreduces the size and firmness of buffalo humps, and reduces truncaladiposity, with no change in peripheral lipodystrophy, while fat freemass increased 5 to 10%. There were no significant or consistent changesin body weight, total body fat, or blood lipids during the treatmentperiod.

Collectively, these clinical studies cited above demonstrate thattherapy with rhGH (SEROSTIM®), administered subcutaneously, in dosesranging from 3 to 6 mg per day for 12 to 24 weeks significantly reducesabnormally accumulated fat, compared to baseline. Specifically,SEROSTIM® (rhGH) has been shown to reduce abdominal girth (Wanke et al,1999), visceral adiposity (Engelson et al, 1999, Mauss et al,1999),buffalo hump (Torres, 1998, 1999), and solitary lipomas (Milano et al,1999). Therapy with rhGH (SEROSTIM®) also increased lean body mass andbody cell mass as quantified by biolectrical impedance analysis (Wankeet al, 1999; Engelson et al, 1999).

Collective side effects included swelling of the fingers or paresthesiadue to tissue turgor, a few transient elevations of fasting glucose andtriglycerides. At 12 weeks, total cholesterol and fasting triglyceridesdropped significantly, while HDL cholesterol and glucose increased, butnone of these changes were deemed clinically significant (Engelson etal, 1999). No additional episodes of hypertension or elevated pancreaticenzymes have been reported.

As noted earlier, additional prospective, randomized, double-blind,placebo-controlled dose-ranging studies are underway to ascertain themost optimally safe and effective dose of SEROSTIM® (rhGH), duration oftreatment, and requirements for periodic re-treatment and/or maintenancetherapy (if any) needed to sustain the reduction of abnormallyaccumulated fat. Other clinical endpoints such as the effects ofSEROTSIM on different adipose tissue depots, lipoprotein profile,insulin sensitivity, early atherosclerotic lesions will also beinvestigated.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

All references cited herein, including journal articles or abstracts,published or unpublished U.S. or foreign patent application, issued U.S.or foreign patents or any other references, are entirely incorporated byreference herein, including all data, tables, figures and text presentedin the cited references. Additionally, the entire contents of thereferences cited within the references cited herein are also entirelyincorporated by reference.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplication such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning an range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

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What is claimed is:
 1. A method for treating HIV-associateddysmorphia/dysmetabolic syndrome (HADDS), comprising administering to apatient in need thereof an effective amount of a substance which ishuman growth hormone (hGH) or a functional derivative, fragment,variant, analog, or salt thereof which retains the biological activityof human growth hormone.
 2. A method in accordance with claim 1, whereinsaid substance is an isolated human growth hormone.
 3. The methodaccording to claim 2, wherein said substance is recombinant human growthhormone.
 4. The method according to claim 1, wherein said substance isadministered subcutaneously.
 5. The method according to claim 1, whereinsaid substance is administered intramuscularly.
 6. The method accordingto claim 1, wherein said HADDS is abnormal visceral adipose tissue (VAT)accumulation and said patient is a HIV/AIDS patient presenting abnormalvisceral adipose tissue accumulation.
 7. The method according to claim1, wherein said HADDS is dorsocervical adipose tissue accumulation(“buffalo hump”) and said patient is a HIV/AIDS patient presentingdorsocervical adipose tissue accumulation (“buffalo hump”).
 8. Themethod according to claim 1, wherein said HADDS is another type ofpathological adipose accumulation associated with HADDS syndromeselected from the group consisting of abnormal accumulation of adiposetissue in submandibular (“horse collar”), supraclavicular, pectoraland/or mammary areas, and/or has lipomas (benign encapsulated fattytumors, either single or multiple), and said patient is a HIV/AIDSpatient presenting with one or more of these abnormal features.
 9. Amethod in accordance with claim 1, wherein said substance is methionylhGH.
 10. A method in accordance with claim 1, wherein said substance is20-K-hGH, a naturally-occurring variant of hGH found in the pituitaryand in the bloodstream.
 11. A method in accordance with claim 1, whereinsaid substance is GH-V, a naturally-occurring variant of hGH found inthe placenta.
 12. A method in accordance with claim 1, wherein saidsubstance is two-chain anabolic protein (2-CAP) formed by controlledproteolysis of hGH with trypsin.
 13. A method in accordance with claim1, wherein said substance is deamidated hGH.
 14. A method in accordancewith claim 1, wherein said substance is sulfoxidated hGH.
 15. A methodin accordance with claim 1, wherein said substance is hGH acetylated atthe N-terminus.
 16. A method for treating the pathological accumulationof adipose tissue in specific regional depots, comprising administeringto a patient in need thereof an effective amount of a substance which ishuman growth hormone or a functional derivative, fragment, variant,analog, or salt thereof which retains the biological activity of humangrowth hormone.
 17. A method in accordance with claim 16, wherein saidsubstance is an isolated human growth hormone.
 18. The method accordingto claim 17, wherein said substance is recombinant human growth hormone.19. The method according to claim 16, wherein said substance isadministered subcutaneously.
 20. The method according to claim 16,wherein said substance is administered intramuscularly.
 21. A method inaccordance with claim 16, wherein said substance is methionyl hGH.
 22. Amethod in accordance with claim 16, wherein said substance is 20-K-hGH,a naturally-occurring variant of hGH found in the pituitary and in thebloodstream.
 23. A method in accordance with claim 16, wherein saidsubstance is GH-V, a naturally-occurring variant of hGH found in theplacenta.
 24. A method in accordance with claim 16, wherein saidsubstance is two-chain anabolic protein (2-CAP) formed by controlledproteolysis of hGH with trypsin.
 25. A method in accordance with claim16, wherein said substance is deamidated hGH.
 26. A method in accordancewith claim 16, wherein said substance is sulfoxidated hGH.
 27. A methodin accordance with claim 16, wherein said substance is hGH acetylated atthe N-terminus.